Abstract Cardiomyopathies (CMs) are group of inherited heterogeneous diseases of heart muscle with no definite effective treatment, ultimately resulting in heart failure (HF), transplant or sudden cardiac death (SCD) in many patients. Despite decades of intense research, it is still difficult to predict CM phenotypes and explain clinical heterogeneity, severity and prognosis. The likely reason for poor genotype-phenotype association is that mutations in single gene do not completely determine disease course; rather interactions of multiple genes, causal and modifier, may be required to explain CM phenotypes. We have screened adult and pediatric patients with various types of CMs, including dilated (DCM), hypertrophic (HCM) and restrictive (RCM) using whole exome sequencing (641 patients) and direct Sanger sequencing (900 patients), and identified myopalladin (MYPN), encoding a cytoskeletal Z-disk protein, as a strong causal gene associated with heterogeneous CM phenotypes with clinical expressions ranging from asymptomatic left ventricular hypertrophy to dilation with progressive HF to SCD or transplant. The CM symptoms are highly varied among individual patients, even within the same family members who carry identical mutations. These variations are influenced by modifier genes that alter the effect of causal gene at major locus. However, modifier genes of MYPN remain largely unidentified and are likely to depend on the interaction of multiple genes in MYPN related gene pathways and gene networks. The identification of modifier genes is now a crucial goal of research in CMs from the viewpoints of diagnosis, treatment and genetic counseling, but it remains very challenging in clinical cohorts. The objective of the current study is to determine modifier genes and molecular networks that modulate severity of MYPN induced CMs using powers of combined reverse and forward genetics and systems genetic analysis. Systems genetics is such an approach to understand complex diseases by focusing on how genes work together in groups rather than singly. We have confirmed that mutation Q529X of MYPN associated with heterogeneous phenotypes in humans causes CM in knock-in mice. We have developed the largest and best characterized mouse genetic reference population (GRP) composed of over 150 lines derived from crosses between C57BL/6J (B6) and DBA/2J (D2) parents. The D2 strain has been identified as mouse CM model, and CM phenotypes from D2 mouse is segregated among the BXD family of strains, which makes BXD family as an excellent platform to identify CM modifiers. Moreover, we have introduced Q526X-Mypn mutation (homologous to human Q529X-MYPN) into BXD genetic background to examine how different genetic background modifies the effect of Mypn mutation on CM phenotypes. This proposal is one of the first multidisciplinary collaborative efforts to identify modifier genes in MYPN induced CM in both human and mouse. Using cross-species validation sources and powerful systems genetics, we will define and validate novel modifier genes that interact with Mypn and are responsible for CM variations.